Alternative fatty acid desaturation pathways revealed by deep profiling of total fatty acids in RAW 264.7 cell line

In-depth structural characterization of lipids provides a new means to investigate lipid metabolism. In this study, we have conducted deep profiling of total fatty acids (FAs) from RAW 264.7 macrophages by utilizing charge-tagging Paternò-Büchi derivatization of carbon-carbon double bond (C=C) and reversed-phase liquid chromatography-tandem mass spectrometry. A series of FAs exhibiting unusual site(s) of unsaturation was unearthed, with their identities being confirmed by observing anticipated compositional alterations upon desaturase inhibition. The data reveal that FADS2 Δ 6-desaturation can generate n-11 C=C in the odd-chain monounsaturated fatty acids (MUFAs) as well as n-10 and n-12 families of even-chain MUFAs. SCD1 Δ 9-desaturation yields n-6, n-8, and n-10 of odd-chain MUFAs, as well as n-5, n-7, and n-9 families of even-chain MUFAs. Besides n-3 and n-6 families of polyunsaturated fatty acids (PUFAs), the presence of n-7 and n-9 families of PUFAs indicates that the n-7 and n-9 isomers of FA 18:1 can be utilized as substrates for further desaturation and elongation. The n-7 and n-9 families of PUFAs identified in RAW 264.7 macrophages are noteworthy because their C=C modifications are achieved exclusively via de novo lipogenesis. Our discovery outlines the metabolic plasticity in fatty acid desaturation which constitutes an unexplored rewiring in RAW264.7 macrophages.

Fatty acid (FA) metabolism consists of anabolic and catabolic processes that are necessary for energy homeostasis as well as the formation of metabolic intermediates required for the maintenance of cell membrane structure and function (1), energy storage (2), and cell signaling (3). The desaturases introduce a carbon-carbon double bond (C=C) at a specific position on the acyl chain, thereby influencing several key biological properties of the fatty acids, including membrane fluidity (4), antioxidant activity (5), and inflammation (6). Mammalian cells express desaturases of Δ 5, Δ 6, and Δ 9 activities, where the Δ− number indicates the position of a C=C counting from the carboxylic acid moiety of FA. The desaturases are classified into two distinct families referred to as stearoyl-coenzyme A desaturase (SCD) (7,8) and fatty acid desaturase (FADS) (9). Cancer cells have an aberrantly activated lipid metabolism that enables them to synthesize, elongate, and desaturate fatty acids to support proliferation (10). Notably, it was recently shown that cancer cells exploit FADS2 to catalyze n-10 C=C formation (i.e., the 10 th carbon counting from the FA methyl terminus) in palmitic acid (FA 16:0) (11), which is usually observed only within lipids from hair and skin (12); however, this desaturation mechanism can be activated to meet the metabolic needs of cancer. Young et al. revealed the promiscuity of human FADS2, which promoted the production of rarely reported n-8, n-10, and n-12 desaturation sites in lipids (13). The studies from the Brenna group showed that FADS2 activity was promiscuous. It can catalyze Δ4, Δ6, and Δ8 desaturation towards at least 16 substrates, including saturated fatty acids, even-and odd-chain monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and branched-chain fatty acids (BCFAs) with chain length ranging from 16 to 24 carbons (14,15). In order to elucidate unexplored plasticity in cancer cells fatty acid metabolism, it is imperative to identify the unsaturation profile of the fatty acyl building blocks. Gas chromatography hyphenated with electron ionization mass spectrometry (GC/EI-MS) is still widely used for profiling of FAs, albeit in the form of fatty acid methyl esters (FAMEs) (16)(17)(18). Identification of FAs relies on matching the retention time of GC-separated FAMEs to those of the standards (16)(17)(18), which inevitably hinders its utility in the discovery of unknown FAs. To overcome this limitation, the Brenna group combined acetonitrile (ACN) chemical ionization (CI) with collision-induced dissociation (CID) for independent structural analysis of unsaturated FAMEs (19). Alternatively, new tandem mass spectrometry (MS/MS) methods, such as ozone-induced dissociation (OzID) (20), ultraviolet photodissociation (UVPD) (21,22), epoxidation (23,24), aziridination (25,26), and the Paternò-Büchi (PB) derivatization (27)(28)(29)(30)(31), have been developed to provide lipidome profiling at the level of C=C positions. For instance, our group recently developed a sensitive and readily adaptable workflow for the quantitation of FAs at C=C location level via chargetagging PB derivatization and reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) (31). This method has advantages in identifying unknown FAs where synthetic standards are not available. It offers a limit of detection (LOD) in the sub-nM range for synthetic standards and relative quantitation of low abundance C=C location isomers (as lows as 1% relative to the most abundant isomer).
In this work, we employ charge-tagging PB derivatization in combination with desaturate inhibition to uncover a diverse set of novel fatty acids with an unusual site(s) of unsaturation in RAW 264.7 macrophages. The latter method allows us to achieve high confidence in the identification even in the absence of authentic standards. Furthermore, desaturate inhibition provides evidence for studying FAs derived from SCD1 Δ9-desaturation and FADS2 Δ6-desaturation activities. Our data suggest that both enzymes display broad activity toward both even and odd-chain saturated FAs and give the cells access to an expanded repertoire of even-chain MUFAs with n-5, n-7, n-9, n-10, and n-12 C=Cs. Furthermore, even-chain MUFAs derived from these de novo synthesized families also act as substrates for further desaturation and elongation, yielding a wide array of PUFAs, consisting of the n-7, n-9, n-10, and n-12 families.

Lipid nomenclature
The shorthand notation recommended by LIPID MAPS consortium was used (32). In brief, the position of C=C in an aliphatic chain is defined by the n-x nomenclature, counting from the methyl terminus. The Z/E stereo-configurations of C=C could not be assigned from PB-MS/MS and thus were not indicated for FAs from biological samples.
RAW 264.7 macrophages (American Type Culture Collection (ATCC); Manassas, VA, USA) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium with 10% fetal bovine serum and 1% Penicillin-Streptomycin solution and collected by centrifugation. The cell pellets were washed, frozen in liquid nitrogen, and stored at −80 • C. For direct inhibition of enzyme activity, RAW 264.7 macrophages were treated for 72 h with 60 μM FADS2 inhibitor (SC26196, purchased from Sigma-Aldrich) and 2.5 μM SCD1 inhibitor (CAY10566, purchased from Sigma-Aldrich), while the control group was treated with DMSO.

Lipid extraction and saponification
The cell samples were extracted using a modified MTBE protocol (33). In brief, MTBE (5 ml), MeOH (1.5 ml), water (1.25 ml), and 1 mM [D4] FA 18:0 (10 μl) were added to the tube containing 2 million cells. The mixture was then vortexed for 5 min. To separate the organic and aqueous phases, the mixture was centrifuged at 10,000 g for 10 min. The upper phase was collected. The lower phase was re-extracted with 2 ml of the upper phase of the system MTBE/MeOH/H 2 O (10/3/2.5, v/v/v). Finally, the combined organic phases were collected and dried under N 2 flow for further use. The cell medium was also subjected to lipid extraction and total FA profiling. The extracted lipids were saponified in 500 μl ACN:15% KOH (50/50, v/v) at 60 • C for 60 min. The solution was acidified with 1M HCl (1 ml). The hydrolyzed total FAs were extracted twice with 1.5 ml isooctane each time. The organic layer was collected, dried under nitrogen, and redissolved in an aliquot of 500 μl MeOH for further derivatization.

AMPP derivatization
AMPP derivatization followed the procedure provided by the vendor (AMP+ MaxSpec Kit). AMPP derivatized sample was extracted twice by 600 μl MTBE and dried under a nitrogen stream. The derivatized sample was resuspended in 100 μl MeOH for RPLC-MS/MS analysis.

Offline 2-acpy PB derivatization of total FAs
The 2-acpy PB derivatization was performed using a homemade flow microreactor (34). Total FA extracts and 10 mM 2-acpy were dissolved in 200 μl ACN. The solution was injected into the flow microreactor for 20 s' UV-irradiation (∼254 nm). About 200 μl reaction solution was collected; The PB derivatized sample was subjected to subsequent RPLC-MS/ MS analyses.

Data analysis and lipid identification
The analysis of RPLC-PB-MS/MS data was carried out using a home-made software, LipidNovelist (35). Data Analysis 5.0 software (Bruker Daltonics) was used to convert .d data to an open file format (.ascii), which can be read by Lip-idNovelist. LipidNovelist conducted de novo analysis of the data generated from RPLC-PB-MS/MS and the C=C location of each unsaturated fatty acid was assigned based on the detection of C=C diagnostic ion pairs. LipidNovelist also calculated the intensity ratios of diagnostic ions corresponding to C=C location isomers for relative isomer quantitation. For relative quantitation at the sum composition level, MS 1 spectra of AMPP-derivatized fatty acid and corresponding internal standards were imported into the LipidNovelist Extension and subjected to Type-I isotope correction. The most up-to-date version of LipidNovelist and LipidNovelist Extension, along with instructional videos and example data to facilitate its utilization, can be accessed at https://doi.org/1 0.6084/m9.figshare.22297771.

Analysis workflow
RAW 264.7 cells have been extensively characterized and are well-suited for lipid metabolism studies due to their expression of a wide range of fatty acid desaturation enzymes involved in lipid metabolism (36)(37)(38). Figure 1 provides an overview of the analysis workflow. After lipid extraction and saponification, [D4] FA 18:0 was added as the internal standard (IS). The total FAs were divided into two equal aliquots. One aliquot underwent AMPP derivatization, while the other aliquot was subjected to 2-acpy derivatization. Subsequently, the AMPP-derivatized FAs were subjected to RPLC-MS analysis. LipidNovelist Extension was used to process the MS 1 data which provided relative quantification (I/I IS ) at the sum composition level (35). The %relative abundance of a certain FA was calculated by normalizing its MS 1 signal to that of all FAs detected in the same RPLC run. Type-I isotope correction was applied to the data prior to relative quanaitation. The associate data are provided in supplemental Table S1.
Then, we used 2-acpy derivatization (the PB reaction) followed by RPLC-MS/MS to achieve relative quantitation of unsaturated total FAs at the C=C location level (31). The PB-MS/MS data were analyzed by the Lip-idOA module (39) embedded in LipidNovelist (35) to achieve de novo identification of FA at the C=C location level. %Relative composition of a specific isomer was calculated by normalizing the relative abundances of the C=C diagnostic ions associated with that isomer to the summed abundances of the diagnostic ions from all C=C location isomers. In order to enhance the accuracy of identification for uncanonical FA C=C location isomers especially in the absence of authentic standards, we utilized SCD1 inhibitor or FADS2 inhibitor to disturb cells and tracked compositional changes of these uncanonical FA C=C isomers. Additionally, the total FAs in the cell medium were profiled and later used to assess the influence of lipid uptake from the environment. To ensure the reliability and robustness of our experimental results, we performed analysis on six biological replicates, each accompanied by three technical replicates. The average coefficient of variation was less than 1% across three technical replicates and less than 8% across six biological replicates.
The retention time (RT) values of AMPP-derivatized FAs were plotted as a function of carbon chain length (14−24) and the number of C=C (0−6) (supplemental Fig. S1). We clearly observe two distinct chromatographic peaks for FA 15:0, FA 17:0, and FA 19:0, respectively. Based on the RT trendline and the literature report (41), the later eluting peaks should correspond to the straight-chain fatty acids, while the earlier eluting peaks might represent their corresponding branched-chain fatty acid isomers. However, even with prolonged separation time (50 min), only a single chromatographic peak was detected for each odd-chain MUFA. Consequently, this investigation was unable to distinguish between straight MUFAs and branchedchain MUFAs.

DISCUSSION
The exploration of FA C=C isomers within RAW 264.7 cell lipidome in combination with enzyme inhibition reveals that cellular fatty acid desaturation is far more complex and dynamic than previously considered. The biosynthesis pathways of the odd-chain MUFAs, even-chain MUFAs, and PUFAs are proposed based on the experimental observations. This expanded network of FA desaturation provides plasticity to generate certain FA families arising from extreme metabolic stress. For instance, the FADS2 plasticity allows for multiple substrate acceptances (e.g., FADS2 Δ 6-desaturation of FA 17:0 to yield FA 17:1 n-11) or an alternative site of desaturation. Similarly, under metabolic stress, SCD1 also displays plasticity toward substrate preference to allow for the formation of n-6, n-8, and n-10 of the odd-chain MUFAs and n-5, n-7, and n-9 of the even-chain MUFA families. The de novo synthesis of n-7, n-9, n-10, and n-12 PUFAs is worth attention, given their structural similarity to the dietary-derived and biologically active FAs, such as arachidonic acid. They may disrupt homeostatic lipid  signaling or fulfill signaling roles in the absence of dietary FA uptake or upon other chemical environment changes. Notably, when de novo lipogenesis of n-7, n-9, n-10, and n-12 of PUFAs is hampered, cells can increase the uptake of dietary FAs (e.g., n-3 and n-6 of PUFAs) to meet metabolic demands. These are consistent with a recent report by the Blanksby group in which hundreds of fatty acid structures were discovered in MCF7 and LNCAP cancer cell lines via OzID (48). In conclusion, this study has revealed various new fatty acids in RAW 264.7 cells with unusual site(s) of unsaturation that are not described by canonical pathways. Although further work is required to specify the biological impact of the unusual lipid unsaturation, the experimental workflows and findings presented in the work can serve as a roadmap toward future discovery.

Data availability
All data concerned with this study are presented within this manuscript.

Supplemental data
This article contains supplemental data.